S205: INHIBITING THE ALARMIN-DRIVEN HEMATOPOIESIS-STROMAL CELLS CROSSTALK IN PRIMARY MYELOFIBROSIS AMELIORATES BONE MARROW FIBROSIS
Authors: Hélène Gleitz, Inge Snoeren, Stijn Fuchs, James Nagai, Aurélien Dugourd, Charlotte Boys, Vanessa Klöker, Jessica Pritchard, Eric Bindels, Peter André Willem Te Boekhorst, Martina Crysandt, Julio Saez-Rodriguez, Thomas Vogl, Rafael Kramann, Ivan Costa, Rebekka Schneider
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that arises from clonal proliferation of hematopoietic stem cells (HSCs) and leads to progressive bone marrow (BM) fibrosis, resulting in extramedullary hematopoiesis (typically in the spleen), BM failure, and ultimately death. Using single-cell RNAseq, we showed disease-specific upregulation of the alarmin complex S100A8/S100A9 in fibrosis-driving mesenchymal stromal cell (MSC) populations (Leimkühler et al., 2021). Importantly, targeting these alarmins with the small molecular oral inhibitor tasquinimod stopped the progression of fibrosis in murine JAK2V617F PMF models. Aims: The questions remained whether the effect of alarmin inhibition of Tasquinimod is more relevant in hematopoietic or stromal cells and how Tasquinimod affects the hematopoiesis-stromal cell crosstalk. Methods: Here, we analyzed the effect of Tasquinimod on hematopoietic cells and stromal cells using RNA sequencing in a JAK2V617F murine PMF model, systematically analyzed the effect of stromal versus hematopoietic S100A9 inhibition using a genetic knockout model, and determined the consequences of S100A9 overexpression using a genetic knock-in model and lentiviral overexpression. Results: Receptor-ligand analysis of RNA sequencing data demonstrated that Tasquinimod indeed targets the disease-specific alarmin-driven interaction between CD41+ megakaryocytes, Lin-Sca1+ stromal cells and CD11b+Gr1- monocytes. The increased alarmin signalling originated from JAK2V617F malignant hematopoietic cells and communicated to stromal cells. In addition, TGFb-based interactions between CD41+ megakaryocytes and Lin-Sca1+ stromal cells were significantly reduced, which shows a direct anti-fibrotic effect of Tasquinimod as TGFb is supposed to be the master switch of fibrosis. Additionally, we observed a significant decrease in PI3K and Myc pro-proliferative signalling in the JAK2V617F mutant hematopoietic cells. In line with this, we functionally demonstrated that tasquinimod specifically induces apoptosis in JAK2V617F cells. We thus concluded that increased alarmin signalling originates from JAK2V617F malignant hematopoietic cells and communicates to stromal cells leading to initiation of fibrosis. We show that only MSCs in co-culture with malignant JAK2V617F cells upregulate alarmins and TGFb and decrease CXCL12 expression, gaining an overall pro-inflammatory and pro-fibrotic phenotype, combined with a loss of hematopoietic support. In line with this conclusion, we confirmed that genetic knockout of S100A8/S100A9 in the hematopoietic compartment, but not in stromal cells, alone ameliorates myelofibrosis, reduces splenomegaly and myeloproliferation in both ThPO- and JAK2V617F-induced PMF. Summary/Conclusion: We demonstrate that alarmins need to be secreted and that transcriptional overexpression by lentiviral overexpression and genetic hematopoietic knock-in of S100A8/S100A9 do not lead to myeloproliferation or to an MPN phenotype as S100A8/S100A9 levels do not increase. The S100A8/S100A9 heterodimer levels determined by ELISA correlate with the MPN severity in murine models but also in large cohorts of patient samples. As a next step, we will answer whether alarmins can act not only as a biomarker for progression but also for tasquinimod response in a clinical trial (tasqForce MPN), which will be started by the end of the year (PIs: P. te Boekhorst, M. Crysandt, Scientific PI: R. Schneider). In murine models, S100A8 levels in the plasma correlate with the response to Tasquinimod making S100A8/S100A9 a putative actionable biomarker. Keywords: Bone Marrow Fibrosis, Bone marrow microenvironment, Myelofibrosis